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A structure-based manual screening and an automatic networking of HPLC–MS/MS data were developed for systematically exploring uridyl peptide antibiotics (UPAs).Twenty-eight UPAs including twenty new structures were illustrated by re-analysis of the Streptomyces sp. SS fermentation broth.Two novel kinds of sansanmycins with 2′,3′-dehydrated 4′,5′-enamine-3′-deoxyuridyl and 5,6-dihydro-5′-aminouridyl were reported for the first time.Sansanmycins (SS), one of several known uridyl peptide antibiotics (UPAs) possessing a unique chemical scaffold, showed a good inhibitory effect on the highly refractory pathogens Pseudomonas aeruginosa and Mycobacterium tuberculosis, especially on the multi-drug resistant M. tuberculosis. This study employed high performance liquid chromatography-mass spectrometry detector (HPLC–MSD) ion trap and LTQ orbitrap tandem mass spectrometry (MS/MS) to explore sansanmycin analogues manually and automatically by re-analysis of the Streptomyces sp. SS fermentation broth. The structure-based manual screening method, based on analysis of the fragmentation pathway of known UPAs and on comparisons of the MS/MS spectra with that of sansanmycin A (SS-A), resulted in identifying twenty sansanmycin analogues, including twelve new structures (1-12). Furthermore, to deeply explore sansanmycin analogues, we utilized a GNPS based molecular networking workflow to re-analyze the HPLC–MS/MS data automatically. As a result, eight more new sansanmycins (13-20) were discovered. Compound 1 was discovered to lose two amino acids of residue 1 (AA1) and (2S, 3S)-N3-methyl-2,3-diamino butyric acid (DABA) from the N-terminus, and compounds 6, 11 and 12 were found to contain a 2′,3′-dehydrated 4′,5′-enamine-3′-deoxyuridyl moiety, which have not been reported before. Interestingly, three trace components with novel 5,6-dihydro-5′-aminouridyl group (16-18) were detected for the first time in the sansanmycin-producing strain. Their structures were primarily determined by detail analysis of the data from MS/MS. Compounds 8 and 10 were further confirmed by nuclear magnetic resonance (NMR) data, which proved the efficiency and accuracy of the method of HPLC–MS/MS for exploration of novel UPAs. Comparing to manual screening, the networking method can provide systematic visualization results. Manual screening and networking method may complement with each other to facilitate the mining of novel UPAs.